Expression of camk2b2. (a) The amino acid sequence of β2 CaMK-II, predicted from its gene sequence (camk2b2) is shown containing only the non-alternative (II and VII) variable region domains. Underlined residues are unique to and therefore diagnostic for β2 CaMK-II. (b) β2 CaMK-II splice variants were RT-PCR amplified from 0 to 84 hpf whole embryos, cloned and sequenced. Four variants were identified based on alternative domain usage; none utilizes the putative nuclear targeting domain III. (c) camk2b2 expression was assessed by in situ hybridization at the 8 and 512-cell, 8- and 16-somite and at indicated hours (24, 34, 36, 48 and 72) post fertilization (hpf). Anterior is to the left except for the head on view of the heart at 36 hpf. Arrow heads indicate fin buds, asterisks indicate heart, v = ventricle, a = atrium, scale bar = 1 mm.

β CaMK-II morphant phenotypes. Embryos are shown at 72 hpf after being injected with either the control mismatch MO (5 ng), camk2b1 MO (5 ng) or camk2b2 MO (2 ng). Scale bar = 1 mm. CaMK-II specific activity (pmol/min/mg) was measured by peptide assays on lysates of 72 hpf embryos injected with the indicated amount of MO.

Tbx5 heart defects can be rescued by CaMK-II. Wild type or cmlc2:DsRed transgenic embryos (k–o) were injected with the mismatch MO (5 ng), camk2b2 MO (2 ng) or tbx5 MO (5 ng) with or without 40 pg wild type δ_C CaMK-II cDNA and imaged at 72 hpf (a–e; k–y) or subjected to cmlc2 in situ hybridization at 48 hpf (f–j). Scale bar = 0.5 mm and anterior is to the left. In f and k, a = atrium and v = ventricle. In p–t, insets reveal bmp4 in situ hybridization patterns in the pectoral fin area. In u–y, brackets indicate extent of pectoral fin length.

Co-localization of camk2b2 with cmlc2. camk2b2 (Cy3, red) or cmlc2 (Alexa 488, green) expression was assessed by in situ hybridization and confocal fluorescent microscopy and then merged in 36 hpf atrial (a–c), 36 hpf ventricular (d–f) and 48 hpf ventricular (g–i) samples.

Early genes responsible for heart and fin specification are expressed in camk2b2 morphants. Gata4 and nkx2.5 expression at the 8-somite stage (a–d), cmlc2 at 21s (e, f) and 24 hpf (g, h), bmp4 at 48 hpf (i, j, m, n) and tbx5 at 48 hpf (o, p) were determined by in situ hybridization for embryos injected with 5 ng camk2 mismatch MO or 2 ng camk2b2 MO. Thin sections of cardiac tissue from mismatch or camk2b2 morphants were stained with methylene blue (k, l). Anterior is to the left in all views.

Tbx5 morphants and mutants decrease CaMK-II levels. Camk2b2 in situ hybridization of 36 hpf embryos injected with 5 ng tbx4 MO (a, b) or 5 ng tbx5 MO (c, d) or uninjected hst siblings (e, f) and hst mutants (g, h) as determined phenotypically. Dorsal views (a, c, e, g) mark fins with arrow heads and head on views (b, d, f, h) mark atrium and ventricle. Scale bar = 0.2 mm. (i) CaMK-II activity was assessed at 72 hpf in at least 5 embryos per condition by CaMK-II peptide assay in the presence of Ca²⁺/CaM. Conditions include uninjected embryos (Uninj), camk2b2 MO A and MO B embryos at 2 ng (b2-A and b2-B), embryos co-injected with 2 ng camk2b2 MO B and 40 pg wild type δ_C CaMK-II cDNA (b2 MO + CaMK), tbx4 and tbx5 morphants (5 ng), hst embryos with (-/-) the hst phenotype (hst mut.) and siblings without (+/-; +/+) the hst phenotype (hst norm.).

Tbx5 promotes β2 CaMK-II Expression. (a) β CaMK-II, δ CaMK-II and actin immunoblots of 10 μg NIH/3T3 lysate from cells which had been transfected with Tbx5-pCDNA3 or empty pCDNA3 vector. (b) CaMK-II specific activity (in pmol/min/mg) and integrated blot densities (normalized) from NIH/3T3 cells were averaged from four separate transfections and are shown with standard deviations. (c) Zebrafish embryos were injected with the indicated amount of pCDNA3 or Tbx5-pCDNA3 and activity assays performed at 72 hpf. Asterisks indicate P < 0.005.